Project Summary Circadian clocks are endogenous oscillators that drive daily rhythms of biological processes. In mammals, circadian clocks are found in the brain and in most peripheral tissues. Together, the distributed clocks constitute the fundamental timing system that coordinates daily behavior, physiology, and metabolism. The mammalian clock is built from a transcriptional feedback loop that generates circadian rhythms at the molecular level. The three PERIOD (PER) and two CRYPTOCHROME (CRY) proteins, transcriptional autoregulatory proteins that are dedicated clock components, form a large nuclear protein complex (PER complex) that lies at the heart of the feedback loop. In recent years our laboratory has used preparative purification to analyze the constituent proteins of the nuclear PER complex, providing new mechanistic insights into its transcriptional actions. In the present application we begin to address the question of how the nuclear PER complex works in an integrated fashion as a macromolecular machine, a challenging but essential next step in understanding. Building on our successful efforts, we propose to characterize the properties, composition, and structural organization of PER complexes from wildtype and mutant mice lacking individual PER or CRY proteins. By uniting preparative purification of PER complexes with mouse genetics and structural biology, the application aims to determine how each PER and CRY protein contributes to the assembly, composition, and three- dimensional structure of the nuclear PER complex. If successful, the project offers to deepen our knowledge of the clock mechanism substantially, possibly to an atomic level of resolution. Advances in understanding the mammalian circadian clock will have important implications for our view of the genetic control of behavior and physiology, as well as for human health and disease. Studies from mouse genetics, human genetics, and occupational health indicate that defects of clock function lead to broad behavioral and metabolic dysfunction, producing, for example, disrupted sleep-wake cycles, abnormal feeding, and a metabolic syndrome closely resembling early-stage diabetes. If successful, the proposed study will provide new insights into genetic and molecular mechanisms controlling fundamental behavioral and physiological programs linked to major diseases.